The tyrosine kinases of the Src family are highly conserved signaling proteins involved in the regulation of cell growth whose catalytic activity can be modulated in response to specific cellular signals. The key role that the Src-family kinases play in the onset of many human diseases, particularly cancer, makes them important targets for therapeutic intervention. All nine members of the Src family are large allosteric multi- domain enzymes formed by a catalytic domain, which is preceded by two peptide binding modules, the Src- homology domains SH2 and SF13. Regulation is affected by three inter-related factors: intramolecular conformational changes, multi-domain reorganization, and intermolecular interactions due to association of signal peptides to binding modules. Phosphorylation of two tyrosines (Tyr527 and Tyr416) has opposing effects on catalytic activity: dephosphorylation of Tyr527 in the C- terminus tail results in the activation of the enzyme, while phosphorylation of Tyr416 which is located in a central activation loop of the kinase domain opens the catalytic site and activates the enzyme. The available crystallographic structures do not, however, explain how the catalytic activity is regulated at the atomic level. To extend our understanding of the factors responsible for the regulation of Src tyrosine kinases at the atomic level, computational models at different levels of details are constructed and used. In addition, experiments are designed to probe the internal dynamics of the multi-domain protein in solution as well as to binding processes playing a key role in Src regulation. Specifically, (1) we will determine and characterize the impact of several factors on the loop-opening activating pathway of the catalytic domain using a novel computational method, (2) we will determine the molecular factors affecting the population equilibrium of the assembly/disassembly process controlling the auto-inhibitory assembled conformation of Src using computations and X-ray solution scattering experiments, and (3) we will characterize quantitatively key interactions involving the SH2 and SH3 domains as a bimolecular and intramolecular association using free energy computations and experiments. The goal of this Revision Application (competitive supplement) is to support the experiments planned in Aim 2 and Aim 3. PUBLIC HEALTH RELEVANCE: We propose a research project to understand the atomic and molecular factors underlying the regulation of "signaling" proteins called Src. Those proteins play a critical role in the onset of many human diseases such as cancer, though how they are controlled at the atomic level is not explained. The research will help better understand the action of drugs targeting Src.